This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Drug resistance is the cellular basis of chemotherapy failure. Identifying novel targets in drug resistance is essential for improving cancer treatment. The overall goals of this project are to examine whether glucosylceramide synthase (GCS) is a novel therapeutic target, and an in vivo efficient oligonucleotide against GCS is a reversal agent for drug resistance. The specific aims include to design a mixed-backbone oligonucleotide (MBO-asGCS) and examine its specificity and efficacy in cells and in tumor-bearing mice, and to characterize its chemo-sensitivity mechanisms. Previous works have showed that MBO-asGCS reverses drug resistance. To characterize its mechanisms, we hypothesize that silencing GCS can restore p53-dependent apoptosis and disrupt cancer stem cells. We found that silencing GCS restored p53 dependent-apoptosis in p53 mutant cancer cells (NCI/ADR-RES, deleted 21 base pairs in exon-5). MBO-asGCS increased the expression levels of wild-type p53, p21 and Bax (down-steam effectors of p53), and drug-induced apoptosis in cells and tumors. Introduction of GCS gene reduced p53-depedent apoptosis. MBO-asGCS restores p53 through ceramide modulated-RNA splicing. We found that GCS overexpression was interrelated to the increase of breast cancer stem cells (BCSCs). The BCSCs with CD44+/CD24-/ESA+ phenotype were increased by 5-fold, and 3-fold in drug-resistant MCF-7/Dox and NCI-ADR/RES cell lines, as compared to MCF-7 cell lines, respectively. In BCSCs, GCS enzyme activity was 2-fold greater than other sorted non-stem cells. Silencing GCS by using MBO-asGCS significantly decreased the numbers of BCSCs in MCF-7/Dox cells. Aggressive tumors were found in all mice inoculated with NCI/ADR-RES and NCI/ADR-RE/GCS cells that were BCSCs-enriched;however, no tumor or metastasis was observed in mice injected with NCI-ADR/asGCS cells. MBO-asGCS significantly decreased the numbers of BCSCs isolated from tumors of NCI-ADR/RES after treatment. These results demonstrate that glycosphingolipids are involved in the regulation of cancer stem cell. Silencing GCS eliminates BCSCs that can reverse drug resistance as well as prevent tumor relapse. With these findings, our further studies will answer how ceramide restores wild-type p53 expression and how GSLs modulate the formation of cancer stem cells.